Establishment Of Agrobacterium-Mediated Genetic Transformation Of Brachypodium Distachyon And Isolation And Expression Analysis Of A Taozr Gene Challenged By Strip Rust Fungus In Wheat

CHAPTERⅠESTABLISHMENT OF AGROBACTERIUM-MEDIATED GENETIC TRANSFORMATION OF BRACHYPODIUM DISTACHYONBrachypodium distachyon is a new model organism as a better alternative to Oryzae sativa,its small physical plant size,a short lifecycle,small genome,and less demanding growth requirements. In addition to, Research on B. distachyon such as, pathogen-host interactions, cytogenetics, mutagenesis, BAC library construction,tissue culture and genetic transformation systems are accumulating. EST studies of B. distachyon support the close evolutionaly relationship with wheat and demonstrate its valuable as a model plant.The effect of medium, carbon source,2,4-D,ecotype, hormone, hygromycin in different concentrations and Agrobacterium different concentrations to induce callus and improve plantlet regeneration and transformation frequency were investigated using immature embryos of Brachypodium distachyon from diploid accessions ABR 6 and hexaploid accessions ABR 102 were as explants. The result showed that the optimal media for callus induction was LS+2,4-D 2.5 mg·L-1+maltose 30 g·L-1+agar 6.5 g·L-1; the media for callus subculture was LS+2,4-D 1.0 mg·L-1+BA 0.2 mg·L-1+maltose 30 g·L-1+ agar 6.5 g·L-1; the differentiation media was LS+KT 0.2 mg·L-1+ CuSO4 0.6 mg·L-1+ maltose 30 g·L-1+ agar 6.5 g·L-1;the rooting media was LS+IAA 0.6 mg·L-1 +maltose 20 g·L-1+ agar 7.0g·L-1. The embryogenic callus, derived from immature embryos of the accession ABR 6 was 65.47% on the basal LS medium. The induction frequency of primary calli was 96.67% on the basic media including 30 g·L-1 maltose. The regenerated rate was 71.25% on the basic media including 0.2 g·L-1 KT. Using diploid Brachypodium distachyon accession ABR 6 as transformation receptor. The most suitable of hpt selective concentration was 40 mg·L-1. The frequency of transformation was 5.0% when the optimum agrobacterium concentration was OD600=0.6. The molecular biology analysis that transgenic plantlets were 7 of 12 and amplified bands in 845 bp(hpt) and 535 bp(mGFP5*).The expression of green fluorescence protein can be observed from leaves of transgenic plants under fluorescence microscope. The transgenic plants were demonstrated further. The experiment optimize the conditions tissue culture and the agrobacterium-mediated transformation system of Brachypodium diatachyon was established. CHAPTERⅡISOLATION AND EXPRESSION ANALYSIS OF A TAOZR GENE CHALLENGED BY STRIP RUST FUNGUS IN WHEATStripe rust, caused by Puccinia striiformis f. sp. Tritici, is one of the most devastating wheat diseases and affect the production and quality in our country. Using the resistance cultivars is the efficient way in controlling the disease. However,analysing the interaction and molecular mechnism between wheat and stripe rust is an important reason to cultivate the resistance cultivars.Using in-silico cloning and RT-PCR methods, a cDNA sequence was isolated from wheat cultivar Suwon 11 infected by strip rust pathogen. Bioinformatics tools were applied to analyse both the cDNA sequence and protein sequence. The expression patterns of the gene in the interaction of wheat and strip rust pathogen, exogenous phytohomones and abiotic stresses,were investigated using real time quantitative(qRT-PCR). The expression levels were evaluate in wheat challenged by Puccinia striiformis f. sp. tritici or abiotic stresses. The wheat OZR gene was obtained from wheat infected by stripe rust pathogen, which was designated TaOZR. Opening Reading Frame(ORF)of TaOZR was 240 bp in length, encoding 80 amino acid with molecular weight 8.67 KD and theoretical pI 9.34. TaOZR containing a signal peptide and a transmembrane might be a secreted protein. TaOZR shared 76% similarity with rice. The transcriptional accumulation of TaOZR was increased in incompatible interaction, and unchanged in compatible interaction. The expression levels of TaOZR were up-regualted by exogenous hormone salicylic acid, ethylene, abascisci acid, jasmonic acid treatments. However, the transcripts of TaOZR had no change in response to various abiotic stresses like low temperature, drought and high salinity. TaOZR gene was firstly cloned and characterized from wheat infected by stripe rust fungus. TaOZR might facilitate wheat defence to stripe rust pathogen through salicylic acid ethylene, abascisci acid, jasmonic acid signal pathways.